Detection of tiopronin in body fluids and pharmaceutical products using red-emissive DNA-stabilized silver nanoclusters as a fluorescent probe.

Tiopronin is a widely used drug for treatment of cystinuria, rheumatoid arthritis and hepatic disorders. It is also an antidote to heavy metal poisoning and a radioprotective agent. A method is described for rapid and sensitive determination of tiopronin using DNA-stabilized silver nanoclusters (DNA-AgNCs) as a fluorescent probe. Tiopronin can selectively bind to DNA-AgNCs to form a stable Ag-S bond upon which the red photoluminescence (best measured at excitation/emission wavelengths of 590/640 nm) is quenched. The finding is used to design an assay that has a linear response in the 1-150 nM tiopronin concentration range and a 270 pM limit of detection. Compared with previously reported methods, the present approach is more rapid, highly sensitive and selective. It has been successfully applied in the detection of tiopronin in spiked urine and serum, and in pharmaceutical products (tablets and injections). Graphical abstract An ultrasensitive and reliable method for tiopronin assay is developed using red-emissive silver nanoclusters as a fluorescent probe. It has been successfully applied in the determination of tiopronin in biological fluids and pharmaceutical products.

Simultaneous determination of tiopronin and its primary metabolite in plasma and ocular tissues by HPLC.

Tiopronin, formally 2-mercaptopropionylglycine (MPG), is currently prescribed to treat cystinuria and rheumatoid arthritis, and its antioxidant properties have led to its investigation as a treatment for cataracts, a condition in which oxidative stress is strongly implicated. To study its accumulation in the eye, a reliable, isocratic HPLC method was developed for the determination of MPG and its primary metabolite 2-mercaptopropionic acid (MPA) in plasma and relevant ocular tissues. This method utilizes pre-column derivatization and fluorescence detection. The 3.5 min separation enables high-throughput analysis, and validation experiments demonstrated that this method is suitable for evaluating ocular accumulation of MPG and MPA at concentrations as low as 66 and 33 nm, respectively. Excellent linearity was achieved over the working concentration range with R2 > 0.997. Extraction recovery was reproducible within each matrix and exceeded 97%. Accuracy was within 13.3% relative error, and intra- and inter-day precisions were within 6% CV and 7% CV, respectively. Sample stability was demonstrated under various storage conditions, and the use of an internal standard conferred exceptional ruggedness. This method has been successfully applied for the determination of MPG and MPA in plasma, cornea, lens and retina following intraperitoneal administration of the drug in Wistar rats.

Determination of tiopronin based on the enhancement of Ru(bpy)5²âº co-reactant electrochemiluminescence.

It was found that tiopronin could strongly enhance the electrochemiluminescence of tris(2,2'-bipyridine) ruthenium(II) (Ru(bpy)3(2+)) in alkaline solution on a bare Pt electrode, based on which a sensitive, simple and rapid method for the determination of tiopronin was established. Under the optimal conditions, the logarithm of ECL enhancement has a linear relationship with the logarithm of tiopronin concentration in the range from 2.0×10(-7) to 2.0×10(-4) mol L(-1) with a detection limit of 1.0×10(-8) mol L(-1) (S/N= 3), and the relative standard deviation of 1.6% (n=7, c=5.0×10(-6) mol L(-1)). The proposed method has been applied to the determination of tiopronin in pharmaceutical preparations and the results were satisfactory with recoveries of 91.7±1.7%, 98.3±1.0% and 100.8±0.5%, respectively, for three different concentration levels (0.61 µmol L(-1), 6.1 µmol L(-1) and 12.2 µmol L(-1)). According to the study of electrochemical behavior, ECL behavior and ECL emission spectrum of Ru(bpy)3(2+)/tiopronin system, a possible ECL mechanism was proposed.

Label-free fluorescence turn on detection of tiopronin with tunable dynamic range based on the ensemble of Alizarin Red S/copper ion.

In this study, a new type of rapid, label-free fluorescence turn-on assay for detection of tiopronin using Alizarin Red S (ARS)/copper ion ensemble is developed. ARS is high fluorescence in BR buffer solution. But, the fluorescence of ARS can be significantly quenched by copper ions due to ground-state complexation. However, in the presence of tiopronin, copper ions were released from the ARS and thus restored the fluorescence of ARS. The assay has several important features. First, the system is simple in design, fast in operation and is more convenient and promising than other methods. Second, the proposed assay eliminated the separation process and sophisticated instrumentations. Third, the detection process can be seen with the naked eye and can be easily adapted to automated high-throughput screening. At last, the assay has high sensitivity and selectivity for tiopronin and the detection limit is 0.8 ng/mL which is lower than or at least comparable to the previous methods. Moreover, the dynamic range of the sensor can be tuned simply by adjusting the concentration of copper ions. Importantly, the protocol offers high selectivity for the determination of tiopronin in pharmaceutical tablets, injection and biological samples with satisfactory results. Thus, the assay shows great potential applications in the fields of pharmaceuticals and clinical analysis.

A microchip electrophoresis strategy with online labeling and chemiluminescence detection for simultaneous quantification of thiol drugs.

An integrated microfluidic device with online labeling and chemiluminescence (CL) detection was developed for the simultaneous quantification of thiol drugs. In this device, the online labeling, electrophoresis separation and CL detection were compactly integrated onto a glass/poly(dimethylsiloxane) (PDMS) hybrid microfluidic chip. CL detection was based on the oxidation reaction of N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and o-phthalaldehyde (OPA) labeled thiol drugs with NaBrO. Four thiol drugs including 2-mercaptopropionylglycine (2-MPG), captopril (CP), 6-thioguanine (6-TG) and 6-mercaptopurine (6-MP) were employed as model compounds to examine the utility of the system. It was indicated that the separation and detection of four drugs can be completed within 90s. Detection limits (S/N=3) for the thiol drugs tested were in the range of 8.9×10(-9)-13.5×10(-9)M. The application of the present system was demonstrated by analyzing the thiol drugs in human plasma samples.

High-sensitivity spectrofluorimetric determination of tiopronin based on inhibition of hemoglobin.

A highly sensitive and simple spectrofluorimetric method for the determination of tiopronin based on its inhibitory effect on the hemoglobin-catalyzed reaction of H(2)O(2) and L-tyrosine was developed. The concentration of tiopronin is linear with decreased fluorescence (ΔF) of the system under the optimal experimental conditions. The calibration graph is linear in the range 1.23 × 10(-8) to 3.06 × 10(-5) mol L(-1) with a detection limit of 6.13 × 10(-9) mol L(-1). The relative standard deviation was 4.38% for 11 determinations of 6.13 × 10(-6) mol L(-1). This method can be used for the determination of tiopronin in pharmaceuticals with satisfactory results.

A highly sensitive fluorescent probe for tiopronin based on the cleavage of 2, 4-dinitrobenzenesulfonate.

A highly sensitive fluorogenic probe for tiopronin was proposed. 2,4-Dinitrobenzenesulfonyl-fluorescein (I) is an almost nonfluorescent compound. Upon mixing with tiopronin in aqueous solution, the 2,4-dinitrobenzenesulfonyl group of I was efficiently removed and its parent dye fluorescein was released, hence leading to dramatic increases in both fluorescence and absorbance of the reaction mixture. Under optimal conditions, the fluorescence increase is linear with tiopronin concentration in the range 5.0-600 ng mL(-1), with a detection limit of 1.5 ng mL(-1) (3sigma). The proposed method has been successfully applied to tiopronin determination in pharmaceutical preparations and in spiked human urine samples.

Spectrophotometric determination of tiopronin using its catalytic reaction between sodium 1,2-naphthoquinone-4-sulfonate and hydroxyl ion.

A novel and simple spectrophotometric method for the determination of tiopronin with sodium 1,2-naphthoquinone-4-sulfonate (NQS) is established in this paper. The detailed mechanism is proposed and discussed. It is based on the fact that tiopronin can catalyze the reaction between sodium 1,2-naphthoquinone-4-sulfonate and hydroxyl ion to form 2-hydroxy-1,4-naphthoquinone in a buffer solution of pH 13.00 at the maximal absorption wavelength of 445 nm. When tetradecyl benzyl dimethyl ammonium chloride (Zeph) is added to the solution, the sensitivity of the reaction is improved. Beer's law is obeyed in a range of 0.39 - 15.67 microg mL(-1). The equation of linear regression is A = 0.11749 + 0.05914C (microg mL(-1)), with a linear correlation coefficient of 0.9973. The detection limit is 0.2 microg mL(-1), RSD is 0.88% and the recovery rate is in the range of 96.6 - 103.9%. Furthermore, the method has been validated and successfully applied to the determination of tiopronin in pharmaceutical samples.

Hepatocyte nuclear factor 4alpha in the intestinal epithelial cells protects against inflammatory bowel disease.

BACKGROUND: Hepatocyte nuclear factor 4alpha (HNF4alpha; NR2A1) is an orphan member of the nuclear receptor superfamily expressed in liver and intestine. While HNF4alpha expression is critical for liver function, its role in the gut and in the pathogenesis of inflammatory bowel disease (IBD) is unknown. METHODS: Human intestinal biopsies from control and IBD patients were examined for expression of mRNAs encoding HNF4alpha and other nuclear receptors. An intestine-specific HNF4alpha null mouse line (Hnf4alpha(DeltaIEpC)) was generated using an Hnf4alpha-floxed allele and villin-Cre transgene. These mice and their control floxed counterparts (Hnf4alpha(F/F)), were subjected to a dextran sulfate sodium (DSS)-induced IBD colitis protocol and their clinical symptoms and gene expression patterns determined. RESULTS: In human intestinal biopsies, HNF4alpha was significantly decreased in intestinal tissues from Crohn's disease and ulcerative colitis patients. HNF4alpha expression was also suppressed in the intestine of DSS-treated mice. In Hnf4alpha(DeltaIEpC) mice, disruption of HNF4alpha expression was observed in the epithelial cells throughout the intestine. In the DSS-induced colitis model Hnf4alpha(DeltaIEpC) mice showed markedly more severe changes in clinical symptoms and pathologies associated with IBD including loss of body weight, colon length, and histological morphology as compared with Hnf4alpha(F/F) mice. Furthermore, the Hnf4alpha(DeltaIEpC) mice demonstrate a significant alteration of mucin-associated genes and increased intestinal permeability, which may play an important role in the increased susceptibility to acute colitis following an inflammatory insult. CONCLUSIONS: While HNF4alpha does not have a major role in normal function of the intestine, it protects the gut against DSS-induced colitis.

Cadmium telluride quantum dots as pH-sensitive probes for tiopronin determination.

The pH-sensitive cadmium telluride (CdTe) quantum dots (QDs) were used as proton probes for tiopronin determination. Based on the fluorescence quenching of CdTe QDs caused by tiopronin, a simple, rapid and specific quantitative method was proposed. Under the optimal conditions, the calibration plot of ln(F(0)/F) with concentration of tiopronin was linear in the range of 0.15-20 microg mL(-1)(0.92-122.5 micromol L(-1)) with correlation coefficient of 0.998. The limit of detection (LOD) (3sigma/k) was 0.15 microg mL(-1)(0.92 micromol mL(-1)). The content of tiopronin in pharmaceutical tablet was determined by the proposed method and the result agreed with that obtained from the oxidation-reduction titration method and the claimed value.

Fluorometric assay of tiopronin based on inhibition of multienzyme redox system.

In this paper, a simple and sensitive fluorimetric method for the determination of tiopronin (N-(2-mercaptopropionyl)-glycine) is proposed. The method is based on the strong inhibitory effect of tiopronin on the multienzyme redox system of hemoglobin, nicotinamide adenine dinucleotide (NADH) and H(2)O(2), in which the intrinsic fluorescence of NADH was employed as the detection signal. The calibration graph is linear in the range 6.13 x 10(-7) to 6.13 x 10(-6) M with a detection limit of 1.65 x 10(-7) M and the relative standard deviation of 2.02%. Kinetics in the pseudo-first-order conditions was investigated by stopped-flow spectrofluorometry and the inhibition mechanism of tiopronin was verified of the competitive type.

A simple and sensitive chemiluminescence method for the determination of tiopronin for a pharmaceutical formulation.

Here we report a rather simple and convenient chemiluminescence (CL) method for the determination of tiopronin. It was based that tiopronin could greatly enhance CL between H2O2 and luminol in a basic alkaline solution. Light emission is intense, and even with a simple setup a high sensitivity could be achieved. The linear range was 3 mM-500 nM with a detection limit of 200 nM. Singlet oxygen and hydroxyl radical were suggested to be produced in this reaction and was responsible for the CL of tiopronin. As a preliminary application, this simple method has been successfully applied into the determination of tiopronin in a pharmaceutical formulation.

Determination of tiopronin in pharmaceuticals using a chemiluminescent flow-injection method.

A flow-injection method for the determination of tiopronin in the range 1 x 10(-7)-7 x 10(-5) M is described. The procedure is based on the chemiluminescent reaction of tiopronin with cerium(i.v.) in sulphuric acid medium using rhodamine 6G and quinine as fluorophors. The flow-injection method is rapid and precise and allows measurements of up to 80 solutions per hour. The applicability of the method to the determination of tiopronin in pharmaceutical preparations was demonstrated by investigating the effect of potential interferences and by analysing commercial preparations.

[New strategy for RNA vectorization in mammalian cells. Use of a peptide vector]. / Nouvelle stratégie pour vectorisation d'ARN dans des cellules de mammifères. Utilisation d'un vecteur peptidique.

A major barrier for gene delivery is the low permeability of nucleic acids to cellular membranes. The development of antisenses and gene therapy has focused mainly on improving methods of oligonucleotide or gene delivery to the cell. In this report we described a new strategy for RNA cell delivery, based on a short single peptide. This peptide vector is derived from both the fusion domain of the gp41 protein of HIV and the nuclear localization sequence of the SV40 large T antigen. This peptide vector localizes rapidly to the cytoplasm then to the nucleus of human fibroblasts (HS-68) within a few minutes and exhibits a high affinity for a single-stranded mRNA encoding the p66 subunit of the HIV-1 reverse transcriptase (in a 100 nM range). The peptide/RNA complex formation involves mainly electrostatic interactions between the basic residues of the peptide and the charges on the phosphate group of the RNA. In the presence of the peptide-vector fluorescently-labelled mRNA is delivered into the cytoplasm of mammalian cells (HS68 human fibroblasts) in less than 1 h with a relatively high efficiency (80%). This new concept based on a peptide-derived vector offers several advantages compared to other compounds commonly used in gene delivery. This vector is highly soluble and exhibits no cytotoxicity at the concentrations used for optimal gene delivery. This result clearly supports the fact that this peptide vector is a powerful tool and that it can be used widely, as much for laboratory research as for new applications and development in gene and/or antisense therapy.

Chemiluminescence determination of tiopronin by flow injection analysis based on cerium(IV) oxidation sensitized by quinine.

A flow injection analysis method is proposed for the determination of tiopronin based upon the oxidation by cerium(IV) in dilute sulfuric acid medium and sensitized by quinine. With the peak height as a quantitative parameter applying optimum working conditions, tiopronin is determined over the 1-400 microM range (150 microliters per injection, n = 10, r = 0.9994) with a detection limit of 0.34 microM and an RSD (n = 10) less than 2% at 20 and 50 microM. The proposed method, combining the advantages of speed and sensitivity, was applied to the routine determination of tiopronin in a pharmaceutical preparation.

Flow-injection fluorimetric determination of penicillamine and tiopronin in pharmaceutical preparations.

Two flow-injection methods for the fluorimetric determination of penicillamine and tiopronin are proposed. The procedures are based on the oxidation of these drugs by thallium(III). In hydrochloric acid medium the fluorescence of thallium(I) formed in the oxidation of penicillamine or tiopronin is monitored using excitation and emission wavelengths of lambda ex = 227 nm and lambda em = 419 nm respectively. Linear calibration graphs were obtained between 3 x 10(-7) and 8 x 10(-6) M for penicillamine and between 8 x 10(-7) and 2 x 10(-5) M for tiopronin with sampling frequencies of 90 and 45 samples h-1 respectively. The relative standard deviations were in the ranges 0.48-0.29% for penicillamine and 1.04-0.31% for tiopronin. The applicability of the method to the determination of both drugs in pharmaceutical preparations was demonstrated by investigating the effect of potential interferences and by analysis of commercial preparations.

Effect of P(i) restriction on renal Na(+)-P(i) cotransporter mRNA and immunoreactive protein in X-linked Hyp mice.

Although renal Na(+)-P(i) cotransporter gene expression is decreased in X-linked Hyp mice, the mutants do respond to P(i) restriction with an adaptive increase in Na(+)-P(i) cotransport maximal velocity in renal brush-border membrane vesicles. In the present study, we examined the mechanism for the adaptive increase in Na(+)-P(i) cotransport in P(i)-deprived Hyp mice and normal littermates, using a cDNA probe encoding a rat, renal-specific Na(+)-P(i) cotransporter (NaPi-2) and a rabbit polyclonal antibody raised against a synthetic NaPi-2-derived peptide. The low-P(i) diet elicited an increase in Na(+)-P(i) cotransport in normal (141 +/- 13 to 714 +/- 158) and Hyp mice (59 +/- 6 to 300 +/- 62 pmol.mg protein-1.6 s-1; means +/- SE, n = 3, P < 0.01) that was accompanied by an increase in brush-border membrane NaPi-2 protein, relative to ecto-5'-nucleotidase, in normal (1.0 +/- 0.1 to 7.6 +/- 1.5) and Hyp mice (0.3 +/- 0.1 to 7.7 +/- 1.4) (means +/- SE, n = 4; P < 0.01). The low-P(i) diet also elicited an increase in the abundance of NaPi-2 mRNA, relative to the 18S RNA, in normal (157 +/- 9% of control diet, P < 0.05) and Hyp mice (194 +/- 10% of control diet, P < 0.01). Immunohistochemistry revealed that NaPi-2 protein was localized to the brush-border membrane of the proximal tubule and that both intensity of the signal and number of immunostained proximal tubules were increased in renal sections from normal and Hyp mice fed the low-P(i) diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of penicillamine or tiopronin in pharmaceutical preparations by flow injection analysis.

Two flow injection analysis (FIA) methods, using spectrophotometric detection, are proposed for the determination of penicillamine or tiopronin [N-(2-mercaptopropionylglycine)]. The procedures are based on the formation of yellow complexes between these thiol-containing drugs and Pd(II), in a 1 M or 0.25 M HCl medium, respectively. With peak height as a quantitative parameter, penicillamine is determined over the range 1.0 x 10(-5)-7.0 x 10(-4) M; for tiopronin the range is 1.0 x 10(-5)-6.0 x 10(-4) M. The methods have been applied to the routine determination of the drugs in pharmaceutical preparations.

A membrane-bound metallo-endopeptidase from rat kidney: its immunological characterization.

The structure and location of a membrane-bound metallo-endopeptidase, previously purified from rat kidney [Yamaguchi et al. (1991) Eur. J. Biochem. 200, 563-571], were examined by immunochemical and immunohistochemical methods with a rabbit polyclonal antibody against the purified enzyme. On treatment with endoglycosidase F, the subunit of the purified enzyme (molecular mass = 88 kDa) was converted to a smaller form (78.5 kDa), indicating that the enzyme contained at least 11% N-linked carbohydrate. Treatment of kidney membranes with papain resulted in release of the enzyme, as shown by Western blotting analysis of the solubilized fraction. Immunoassays of rat tissues showed that only the kidney, and small and large intestine expressed significant amounts of the antigen. Moreover, immunohistochemical studies showed that the antigen was confined to the luminal surfaces of the proximal renal tubules and the intestinal villi. Thus, like another kidney membrane metallo-endopeptidase, meprin [Kounnas et al. (1991) J. Biol. Chem. 266, 17350-17357], the purified enzyme is shown to be a glycoprotein that is probably anchored in the plasma membrane, and located in the luminal surface of microvillar membranes of the kidney and intestine. These results indicate that our enzyme and meprin have clear structural and topological similarities.